Epstein-Barr virus (EBV) infection in humans is associated with the development of both lymphoid and epithelial cell malignancies. EBV can infect cells in either a "latent" or lytic form. Transformation of cells by EBV is associated with the latent form of infection. EBV infection of normal epithelial cells in vivo occurs only in differentiated cells and is completely lytic. In contrast, EBV infection in the epithelial tumor, undifferentiated nasopharyngeal carcinoma (NPC), is primarily latent. Lytic EBV infection is mediated by the expression of the EBV immediate- early (IE) protein, BZLF1 and BRLF1. The BZLF1 protein activates the switch from latent to lytic infection in B cells. However, in epithelial cells, we have recently shown that expression of the BRLF1 immediate- early protein can induce the switch from latent to lytic infection in vitro. Similarly, the BZLF1 protein can also induce lytic viral replication in epithelial cells. The transcriptional control of the BZLF1 and BRLF1 promotes by cellular proteins thus plays an essential role in modulating the stringency of viral latency. Our preliminary data indicate that epithelial cell differentiation activates both the BZLF1 and BRLF1 promoters, suggesting that the lack of BZLF1/BRLF1 transcription in NPC could reflect the undifferentiated state of these tumors. Conversely, we have recently shown that the EBV IE proteins regulate epithelial cell differentiation, as well as cell cycle progression. We hypothesize that regulation of epithelial cell differentiation and cell cycle progression by EBV IE proteins is required for maximal efficiency of lytic replication in vivo. In this grant, we propose to continue our long-term studies investigating the mechanisms by which EBV latency is maintained or disrupted, with particular emphasis upon understanding the biology of EBV infection in epithelial cells. Our specific aims are the following: 1) We will identify the cellular protein(s) which activate the BZLF1 and BRLF1 promoters during epithelial cell differentiation in vitro. 2) We will further explore the mechanism(s) by which the BRLF1 gene product disrupts viral latency in epithelial cells in vitro, and determine if either BZLF1 or BRLF1 gene product disrupts viral latency in epithelial cells in vitro, and determine if either BZLF1 or BRLF1 expression is sufficient to activate lytic viral infection in undifferentiated NPC in vivo. 3) We will define the effects of the BZLF1 and BRLF1 immediate- early proteins upon epithelial cell differentiation and cell cycle regulation, and determine if these effects are important for disruption of viral latency in transient transfection experiments. The proposed studies should help define why EBV infection is normally lytic in epithelial cells (but not B cells), and will explore the mechanisms by which EBV infection becomes latent during the development of NPC.